Mitochondrial biology. Replication-transcription switch in human mitochondria

Abstract

Coordinated replication and expression of the mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mitochondrial DNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase and nascent transcript prevents the generation of replication primers and increases transcription processivity and thereby serves as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as a consequence, respiration and adenosine triphosphate production without the need to replicate mitochondrial DNA, as has been observed during spermatogenesis and the early stages of embryogenesis.

Figure 1. TEFM prevents termination at CSBII

(A) Schematic illustration of the human mtDNA region downstream of LSP promoter. The numbers below the scheme corresponds to the reference human mtDNA; the transcribed sequence of CSBII is shown at the top. (B) MtRNAP does not terminate at CSBII in the presence of TEFM. Termination efficiency (%) is indicated below the panel. (C) A rare polymorphism in a reference mtDNA results in a decreased efficiency of transcription termination at CSBII.

Figure 2. Interactions of TEFM with the components of the elongation complex

(A) TEFM interacts with the RNA in a scaffold EC. Top panel: cross-linking of labeled RNA to the proteins indicated; bottom panel, RNA extension. (B) TEFM interacts with the downstream DNA. (C) Putative region of interaction with TEFM. Structure of human mtRNAP EC (surface representation, PDB ID 3BOC) is shown with the major domains indicated (fingers - pink, palm - light green, N-terminal domain - dark green, thumb - orange, DNA template strand - blue, non-template strand - cyan, RNA - red). The 9 nt long RNA in the EC is extended by 9 nt to model the trajectory of the nascent RNA.

Figure 3. TEFM increases processivity and stability of mtRNAP EC

(A,B) TEFM increases the EC processivity. Transcription was performed using LSP templates lacking CSBII sequence to generate 500 nt (A) or 1000 nt (B) run-off products. Efficiency of synthesis of 500 nt RNA in the presence of TEFM is taken as 100%. (C) TEFM increases the EC stability. Complexes halted at +35 in the presence or absence of TEFM were incubated for the times indicated and chased with CTP. Numbers below indicate efficiency of a halted EC extension (%).

Figure 4. Model for a replication-transcription switch in mitochondria

In the absence of TEFM, mtRNAP initiates transcription at the LSP promoter but terminates at CSBII and produces a 120 nt replication primer at the replication origin oriH. The primer is used by replisome for replication of the heavy strand of mtDNA during which a separate initiation event involving mtRNAP generates a replication primer at oriL. Due to low processivity, transcription from the HSP promoter does not produce a full-size transcript (bottom left). When TEFM is bound, mtRNAP cannot terminate at CSBII and generates full-size transcripts from both LSP and HSP promoters (bottom right). TEFM antitermination activity and its effects on EC processivity ensures that replication events do interfere with the expression of mitochondrial genome.